The overall goal of the project is to use directed proteomics experiments to elucidate the protein-protein interactions mediated by the LDL-receptor-related protein (LRP1), the amyloid precursor protein (APP) and their interacting partners in both the intracellular and extracellular compartment. Mutations within LRP1 have been shown to affect APP processing. Our hypothesis is that protein-protein interaction events connect ligand binding, LRP1, and APP and these events control the production of Aa. Our goal is to use proteomics to define these protein-protein interactions in order to better understand the disease-related aging events that produce Abeta. Both intracellular and extracellular events have been implicated in AD and we plan to probe both of these environments, using both intracellular and extracellular domains of LRP1 and APP in our experiments. Both LRP1 and APP are expected to form multiprotein complexes. Biacore (surface plasmon resonance, SPR) experiments will be utilized to determine the order of binding and which proteins in the complexes interact directly. Amide hydrogen exchange MALDI-mapping experiments will be utilized to identify the protein-protein interface. Brain lysates that are age correlated as well as lysates from both healthy and Alzheimer's diseased brains will be monitored and interacting partners will be quantified using isotope labeling strategies (ICAT). The specific aims of the proposal are: 1) Using peptide-directed proteomics, we will determine which proteins bind to the intracellular region of LRP 1. After identification of the binding proteins, Biacore/surface plasmon resonance (SPR) will be used to determine which proteins in the complexes interact directly. Finally, the binding sites of each protein to their binding partners will be mapped using MALDI-Mapping. 2) Using tagged extracellular regions of LRP 1, we will find LRP 1 binding partners in the secreted proteome. The extracellular proteins that bind to LRP 1 will be used to treat cells and the amount of Aa produced will be monitored. 3) Using peptide-directed proteomics, we will determine which proteins bind to the intracellular region of the amyloid precursor protein (APP). As in Aim 1, Biacore/SPR will be used to determine which proteins interact directly and these will be subjected to MALDI-Mapping. 4) Quantitative proteomics (ICAT) will be applied to age-correlated, AD and healthy brain lysates to develop a correlation between amounts of APP and LRP 1-protein interaction complexes and Abeta production.